Fig 2: Overexpression of Rit2 does not affect overall autophagic flux.a The autophagic flux was assessed in G2019S-LRRK2, G2019S-LRRK2 + Rit2 and G2019S-LRRK2 + GFP cells upon treatment with CQ (100 µM, 3 h) and WB for LC3B. b The ratio between LC3B-II and LC3B-I was not different, suggesting no differences in autophagic flux (n = 3). c Quantification of LC3B levels (normalized to β-actin) indicated no differences upon Rit2 overexpression or CQ-treatment (n = 3). d CytoID assay was employed to visualize autophagosome and autolysosome distribution. e Quantification of CytoID-positive puncta revealed a significant increase in G2019S-LRRK2 cells, when Rit2 was overexpressed (n = 4). Data are represented as median, boxes show the IQ and whiskers show min–max or means ± SEM. In imaging experiments, analysis was conducted on 700–1000 cells per group in each experiment. ***p < 0.01, unpaired two-tailed Student’s t test.

Fig 3: Enhanced Rit2 expression reduces total aSyn and pS129-aSyn levels.a Total aSyn levels in mice injected with AAV-A53T-aSyn alone or in combination with AAV-Rit2 were assessed by blotting for total and phosphorylated aSyn and β-actin. b Quantification of total aSyn levels, normalized on β-actin (n = 4). Co-injection of AAV-Rit2 significantly reduces aSyn levels in the ipsilateral side. c Quantification of pS129-aSyn levels, normalized on β-actin (5 animals/group). Co-injection of AAV-Rit2 significantly reduces pS129-aSyn levels in the ipsilateral side. d Quantification of pS129-aSyn levels, normalized on total aSyn. Co-injection of AAV-Rit2 does not alter pS129-aSyn/aSyn ratio. e IHC staining of pS129-aSyn and TH in the midbrain of AAV-GFP, AAV-A53T-aSyn and AAV-A53T-aSyn + AAV-Rit2 injected mice. f Quantification of pS129-aSyn intensity. AAV-A53T-aSyn injection significantly increases the intensity of pS129-aSyn signal, which is reduced by the co-injection of AAV-Rit2 (5 animals/group). Scale bar = 20 um. Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by the Bonferroni’s post hoc test.

Fig 4: Rit2 KD leads to abnormal lysosomal morphology and proteolytic activity in primary dopaminergic neurons.a Lysosomes were visualized with the Lysotracker Red dye in neurons. b The number of lysosomes per cell was quantified and revealed a decrease when Rit2 levels were reduced (n = 3). c The average size of lysosomes was assessed, and a significant increase of the diameter was measured upon Rit2 KD (n = 3). d The DQ-Red-BSA assay was employed to assess the proteolytic activity of lysosomes. e, f Quantification of DQ-Red-BSA fluorescent spots revealed a significant decrease of number and intensity in Rit2 KD neurons (n = 3). Data are represented as median, boxes show the IQ and whiskers show min–max. *p < 0.05, ***p < 0.001, unpaired two-tailed Student’s t test.

Fig 5: Lysosomal morphology and proteolytic activity are altered in Rit2 KO neuroblastoma cells.a CytoID assay was employed to visualize autophagosome and autolysosome distribution. b Quantification of CytoID-positive puncta revealed a significant increase in Rit2-KO cells (n = 3). c Cell processing with the Lysotracker Red dye was performed to visualize number and size of lysosomes. d The number of lysosomes per cell was quantified and revealed a decrease, with Rit2 KO (n = 6). e The average size of lysosomes was assessed, and a significant increase of the diameter was measured in Rit2-KO cells (n = 6). f The DQ-Red-BSA assay was employed to assess the proteolytic activity of lysosomes. g, h Quantification of DQ-Red-BSA fluorescent spots revealed a significant decrease of number and intensity in Rit2-KO cells (n = 3). Data are represented as median, boxes show the IQ and whiskers show min–max. In imaging experiments, analysis was conducted on 700–1000 cells per group in each experiment. *p < 0.05, ***p < 0.001, ****p < 0.0001 unpaired two-tailed Student’s t test.